Genetic diversity based on multivariate techniques in elephant grass genotypes for bioenergy.

The use of biomass for energy production constitutes a promising strategy that warrants the search for new sources of biomass. Elephant grass has been gaining notoriety due to its high dry matter yield and rapid growth. The present study was carried out to quantify the genetic divergence of nine elephant grass half-sib families in order to identify genotypes with greater genetic divergence and productive potential for hybridization, using the hierarchical clustering methodology based on principal components. Half-sib families were generated using genotypes from the Active Germplasm Bank of Elephant Grass. The experiment was laid out in a randomized-block design with nine half-sib families, three replicates, and eight plants per plot. A total of 216 genotypes of elephant grass were evaluated. Principal component (PC), biplot, and hierarchical clustering analyses for diversity estimation were conducted using R software. The first two PCs of biplot analysis accounted for 64% of the cumulative variation. Dry matter yield was the most important trait for genotype discrimination (0.89), followed by plant height (0.67) and stem diameter (0.61) in PC1. In this analysis, the distances between accessions were considered and there were no family links, which indicates the existence of wide variability within the evaluated families, since genotypes belonging to the same family were not grouped together, but rather distributed into different groups. Crosses between genotypes of group three and genotypes of groups one and two are recommended for the development of high-yielding genotypes when aiming at energy production.

ForMAX - a beamline for multiscale and multimodal structural characterization of hierarchical materials.

The ForMAX beamline at the MAX IV Laboratory provides multiscale and multimodal structural characterization of hierarchical materials in the nanometre to millimetre range by combining small- and wide-angle X-ray scattering with full-field microtomography. The modular design of the beamline is optimized for easy switching between different experimental modalities. The beamline has a special focus on the development of novel fibrous materials from forest resources, but it is also well suited for studies within, for example, food science and biomedical research.

Characterizing pregnancy losses in Bos indicus beef females receiving a fixed-timed artificial insemination protocol.

This experiment evaluated pregnancy losses from day 30 of gestation to calving in Bos indicus females, and evaluated if serum haptoglobin concentrations during early gestation impacts subsequent pregnancy losses. A total of 4926 Nelore females were used, being 1802 nulliparous heifers inseminated as yearlings (precocious heifers), 1356 nulliparous heifers inseminated at 24 months of age (conventional heifers), 887 suckling primiparous cows, and 881 suckling multiparous cows. Cows were assigned to an ovulation synchronization + fixed-time artificial insemination (FTAI) protocol from day -11 to 0 of the experiment. Pregnancy status was verified using transrectal ultrasonography on days 30 and 60 after FTAI, via transrectal palpation on day 150 after FTAI, and according to calf birth. Blood samples were collected from all animals diagnosed pregnant on day 30 after FTAI, and analyzed for serum concentrations of haptoglobin and pregnancy associated glycoproteins (PAG). Pregnancy loss was greater (P < 0.01) from day 60-150 of gestation (10.2 %) compared with day 30-60 of gestation (6.0 %) and with day 150 of gestation to calving (7.4 %), and differed (P = 0.04) between these latter two periods. Pregnancy loss from day 30-60 of gestation did not differ (P = 0.26) among parities, whereas total pregnancy losses (day 30 to calving) were greater (P < 0.01) in precocious (28.4 %) and conventional (27.1 %) heifers compared with primiparous (16.4 %) and multiparous (13.0 %) cows. Serum PAG concentrations on day 30 after FTAI were less (P ≤ 0.03) in cows that lost the pregnancy (5.63 ng/mL) from day 30-60 of gestation, as well as those that lost the pregnancy from day 30 to calving (8.59 ng/mL) compared with cohorts that maintained the pregnancy (9.39 and 9.32 ng/mL, respectively). No differences in serum PAG concentrations on day 30 after FTAI were noted (P ≥ 0.23) according to pregnancy losses from day 60 to calving. Serum haptoglobin concentration on day 30 after FTAI also did not differ (P ≥ 0.48) between cows that maintained or lost the pregnancy. This experiment provides novel information about pregnancy losses after day 30 of gestation in B. indicus cattle, with most losses occurring as fetal mortality and not affected by systemic inflammation during early gestation. Pregnancy losses were nearly doubled in precocious and conventional heifers compared with parous cows, demonstrating the relevance of this reproductive failure to B. indicus replacement heifers.

Effects of mixtures of herbicides on nutrient cycling and plant support considering current agriculture practices.

The use of mixtures of pesticides and consecutive pesticide applications challenge current regulations aimed at protecting ecosystem health due to unpredictable effects of complex and dynamic mixtures. In this study, we tested the ecotoxicological effects of mixtures of herbicides, applied following a real application scheme of soybean production on soil health in a mesocosm experiment. The experiment included two sequential applications; first, glyphosate + dicamba + clethodim, and 30 days later, flumioxazin + metolachlor. Commercial products were used at the recommended doses and at two other concentrations: half and double the recommended dose. Soybean plants were exposed to the herbicide-contaminated soil from the time of sowing to the beginning of pod formation. Half of the plants were harvested at the vegetative stage and the remaining plants at the reproductive stage to evaluate endpoints related to plant support and nutrient cycling. Plant biomass was significantly affected during the vegetative stage at the recommended and double the recommended dose, with the effects being mixture-dose dependent. Lower total and arbuscular colonization of mycorrhizas were also observed in double the recommended dose, and intermediate results were observed for the recommended dose. Nodule mass and phosphorous concentration in plants decreased with increasing herbicide doses. By the end of the experiment, nodule mass and total mycorrhizal colonization were low in the plants treated with double the recommended dose of herbicides. However, both endpoints reached similar values to the control at lower herbicide doses. Plant height and phenology were only lower at double the recommended dose during the experiment. The use of non-standard endpoints evidenced that important soil functions were transiently or permanently affected, while the realistic application scheme accounted for the impact of the management practice currently used. Pesticide risk assessment should therefore, incorporate both issues to effectively protect the ecosystems.

Clinical and morphological features of large-cell neuroendocrine carcinomas and small-cell lung carcinomas expressing the DLL3 and ASCL1 oncoproteins.

Intratumoral similarities and differences between large-cell neuroendocrine carcinomas (LCNECs) and small-cell lung carcinomas (SCLCs) are determined partially by the Notch signaling pathway, which controls the switch from neuroendocrine to slight/non-neuroendocrine cell fate. LCNECs are divided into two subgroups according to genomic alterations: type I LCNECs exhibit a neuroendocrine profile characterized by achaete-scute homolog 1 (ASCL1)high/delta-like protein 3 (DLL3)high/NOTCHlow and type II LCNECs show the pattern ASCL1low/DLL3low/NOTCHhigh. Here, we used immunohistochemistry, transmission electron microscopy, and digital analysis to examine the role of the Notch ligand DLL3 as an immunomarker of the neuroendocrine state and ASCL1 as a regulator of cell-cell interactions in SCLCs and LCNECs. High DLL3 and ASCL1 expression was associated with atypical submicroscopic characteristics involving nuclear size, chromatin arrangement, Golgi apparatus, and endoplasmic reticulum, and was characteristic of type I LCNECs with similarity to SCLCs, whereas low DLL3 and ASCL1 expression was found in both SCLCs and type II LCNECs. In patients diagnosed at an early stage who did not have metastasis and who underwent chemotherapy, DLL3high and ASCL1high SCLCs and type I LCNECs were associated with a better prognosis and a lower risk of death. The present findings suggested that DLL3/ASCL1 are potential therapeutic targets and prognostic indicators in patients with SCLCs or LCNECs.

Improved separation and quantification method for microplastic analysis in sediment: A fine-grained matrix from Arctic Greenland.

Microplastic analysis requires effective separation and purification methods, which greatly depend on the matrix and target particle size. Microplastics-sediment extraction usually involves intermediate steps, increasing processing time and particle loss, particularly for particles <100 µm. Here, we propose an improved separation and quantification method for fine-grained sediment that minimizes microplastic loss by reducing intermediate steps. First, the sample is treated with CH3COOH, KOH and NaClO, and only transferred for the density separation (ZnCl2). The extraction efficiency, visually evaluated on spiked samples, was higher than 90% for particles >100 µm and 83% for 63-75 µm particles. This indicates that a sequential extraction method reduces the risk of particle loss, particularly of the small size fraction. Comparatively, the extraction of ABS particles (20-100 µm) was low (30%) but the recovery, assessed via µFTIR, was higher (55%). Additionally, the proposed method can be adapted to other sediment types and environmental matrices.

Recombinant human TIM-3 ectodomain expressed in bacteria and recovered from inclusion bodies as a stable and active molecule.

Introduction: Microbial systems, such as Escherichia coli, as host recombinant expression is the most versatile and the cheapest system for protein production, however, several obstacles still remain, such as recovery of soluble and functional proteins from inclusion bodies, elimination of lipopolysaccharides (LPS) contamination, incomplete synthesis, degradation by proteases, and the lack of post-translational modifications, which becomes even more complex when comes to membrane proteins, because they are difficult not only to produce but also to keep in solution in its active state. T-cell Immunoglobulin and Mucin domain 3 (TIM-3) is a type I transmembrane protein that is predominantly expressed on the surface of T lymphocytes, natural killer (NK) cells, dendritic cells, and macrophages, playing a role as a negative immune checkpoint receptor. TIM-3 comprises a single ectodomain for interaction with immune system soluble and cellular components, a transmembrane domain, and a cytoplasmic tail, responsible for the binding of signaling and scaffolding molecules. TIM-3 pathway holds potential as a therapeutic target for immunotherapy against tumors, autoimmunity, chronic virus infections, and various malignancies, however, many aspects of the biology of this receptor are still incompletely understood, especially regarding its ligands. Methods: Here we overcome, for the first time, the challenge of the production of active immune checkpoint protein recovered from bacterial cytoplasmic inclusion bodies, being able to obtain an active, and non-glycosylated TIM-3 ectodomain (TIM-3-ECD), which can be used as a tool to better understand the interactions and roles of this immune checkpoint. The TIM-3 refolding was obtained by the association of high pressure and alkaline pH. Results: The purified TIM-3-ECD showed the correct secondary structure and was recognized from anti-TIM-3 structural-dependent antibodies likewise commercial TIM-3-ECD was produced by a mammal cells system. Furthermore, immunofluorescence showed the ability of TIM-3-ECD to bind to the surface of lung cancer A549 cells and to provide an additional boost for the expression of the lymphocyte activation marker CD69 in anti-CD3/CD28 activated human PBMC. Discussion: Taken together these results validated a methodology able to obtain active checkpoint proteins from bacterial inclusion bodies, which will be helpful to further investigate the interactions of this and others not yet explored immune checkpoints.

Symptoms of Long-COVID 1-Year after a COVID-19 outbreak among sailors on a French aircraft carrier.

OBJECTIVES: While persistent symptoms have been reported after the coronavirus disease-2019 (COVID-19), long-term data on outpatients with mild COVID-19 are lacking. The objective was to describe symptoms persisting for 12 months. METHODS: This prospective cohort study on 1767 sailors of an aircraft carrier in which a Covid-19 outbreak occurred during a mission in April 2020 described predefined self-reported symptoms of Long-COVID at 6, 9 and 12 months. Logistic-regression analyses were used to identify correlates for Long-COVID at months 6, 9 and 12. RESULTS: Among the 641 participants, 619 (35%) completed at least one follow-up questionnaire (413 COVID-positive and 206 COVID-negative). Symptoms of Long-COVID were reported by 53.7%, 55.2% and 54.3% of COVID-positive participants vs 31.2%, 23.3% and 40.0% in COVID-negative patients, at 6 (p <.002), 9 (p <.002) and 12 months (p =.13), respectively. The most frequent symptoms reported were concentration and memory difficulties, asthenia and sleep disorders. CONCLUSION: In this study more than half of COVID-positive outpatients reported persistent symptoms up to 12 months post-quarantine. These findings suggests that all patients, including those with mild disease, can be affected by Long-COVID. A lack of difference at 12 months with COVID-negative patienys prompts caution. The symptoms of Long-COVID are so non-specific that they may be viewed as the consequence of multiple intercurrent factors.

Influence of the river flow pulse on the maturity, resilience, and sustainability of tropical coastal ecosystems.

Coastal marine ecosystems have structural and functional features usually connected by the seasonal transfer of nutrients and organisms. These environments can utilize inter-ecosystem subsidies to increase resilience and maturity and support human activities like fishing. However, the importance of the connection and the role of the seasonal pulse of energy flows to enhance maturity are still poorly understood and reported. Our objective in this paper is to assess the effect of seasonal hydrological pulses on two tropical coastal interconnected ecosystems. Thus, we made four Ecopath models for estuarine and neritic environments considering the dry and rainy seasons, with a similar sampling design that allowed them to be compared. Our results provide evidence for the occurrence of the pulsed ecosystems since both environments seem driven by the river flow. Estuary presents more and more substantial differences (measured by ecosystem attributes) in both seasons because it is directly affected by river floods than the neritic environment. The neritic is affected indirectly by the movement of species from the estuary and by a weaker river flow. In the dry season, the differences between ecosystems are lower because the dry season trend to homogenize cycling, maturity, homeostasis, and resilience. We found that the seasonal river flow (pulse) forces the variability of biomass, flows, and ecosystem features, and this variance creates the required stability for both ecosystems. Still, these environments benefit through the exchange of components that relieve the pressures of predation on specific groups and maintain the energy flow necessary for the functioning of their trophic webs. The pulse by the rainfall favors connectivity and equalizes the two systems, increasing the connectivity between them and the exchange of subsidies that strengthens the trophic structures, contributing to the increase in maturity. In these ecosystems, seasonal changes become a key factor for exchanging flows that will promote sustainability, the accumulation of more biomass (growth), and the optimization of reserve energy (development) in both systems. This efficient joint strategy of perpetuation is what promotes resistance and resilience to these ecosystems, which together can reach different states of equilibrium, translated into maturity to withstand new environmental changes.

Integrative analysis in toxicological assessment of the insecticide Malathion in Allium cepa L system / Análise integrativa na avaliação toxicológica do inseticida Malation no sistema Allium cepa L

For many centuries human populations have been suffering and trying to fight with disease-bearing mosquitoes. Emerging and reemerging diseases such as Dengue, Zika, and Chikungunya affect billions of people around the world and recently has been appealing to control with chemical pesticides. Malathion (MT) is one of the main pesticides used against mosquitoes, the vectors of these diseases. This study aimed to assess cytotoxicity and mutagenicity of the malathion for the bioindicator Allium cepa L. using a multivariate and integrative approach. Moreover, an appendix table was compiled with all available literature of insecticides assessed by the Allium cepa system to support our discussion. Exposures during 48h to 0.5 mg mL-¹ and 1.0 mg mL-¹ MT were compared to the negative control (distilled water) and positive control (MMS solution at 10 mg L-¹). The presence of chromosomal aberrations, micronuclei frequency, and mitotic index abnormalities was evaluated. Anaphase bridges were the alterations with higher incidence and presented a significantly elevated rate in the concentration of 0.5 mg mL-¹, including when compared to the positive control. The integrative discriminant analysis summarizes that MT in assessed concentrations presented effects like the positive control, corroborating its potential of toxicity to DNA. Therefore, it is concluded that MT in its pure composition and in realistic concentrations used, has genotoxic potential in the biological assessment of A. cepa cells. The multivariate integrative analysis was fundamental to show a whole response of all data, providing a global view of the effect of MT on DNA.

Por muitos séculos, as populações humanas sofrem e tentam combater os mosquitos transmissores de doenças. Doenças emergentes e reemergentes como Dengue, Zika e Chikungunya afetam bilhões de pessoas em todo o mundo e, recentemente, vem apelando ao controle com pesticidas químicos. O Malation (MT) é um dos principais pesticidas usados contra mosquitos, vetores dessas doenças. O objetivo deste estudo foi avaliar a citotoxicidade e a mutagenicidade do MT para o bioindicador Allium cepa L. usando uma abordagem multivariada e integrativa. Além disso, uma tabela suplementar foi compilada com toda a literatura disponível de inseticidas avaliada pelo sistema Allium cepa para apoiar nossa discussão. Exposições ao MT durante 48h a 0,5 mg mL-¹ e 1,0 mg mL-¹ foram comparadas a um controle negativo (água destilada) e um controle positivo (10 mg L-¹ de MMS). Foram avaliadas a presença de aberrações cromossômicas, frequência de micronúcleos e anormalidades no índice mitótico. As pontes anafásicas foram as alterações com maior incidência e apresentaram uma taxa significativamente elevada na concentração de 0,5 mg mL-¹, inclusive quando comparadas ao controle positivo. A análise discriminante integrativa resume que o MT nas concentrações avaliadas apresentou efeitos semelhantes ao controle positivo, corroborando seu potencial de toxicidade para o DNA. Portanto, conclui-se que o MT, em sua composição pura e nas concentrações realistas utilizadas, possui potencial genotóxico na avaliação biológica de células de A. cepa. A análise integrativa multivariada foi fundamental para mostrar uma resposta completa de todos os dados, fornecendo uma visão global do efeito da MT no DNA.

Variations in heterochromatin content reveal important polymorphisms for studies of genetic improvement in garlic (Allium sativum L) / Conteúdo de heterocromatina revela polimorfismos importantes para o melhoramento genético do alho (Allium sativum L)

Allium sativum L. is an herb of the Alliaceae family with a specific taste and aroma and medicinal and nutraceutical properties that are widely marketed in several countries. Brazil is one of the largest importers of garlic in the world, despite of its production is restricted and limited to internal consumption. Thus, explore the genetic diversity of commercial garlic conserved at germplasm banks is essential to generate additional genetic information about its economically important crop. A suitable tool for this purpose is the cytogenetic characterisation of these accessions. This study aimed to characterise the cytogenetic diversity among seven accessions of garlic from a Germplasm Bank in Brazil. The karyotypes were obtained by conventional staining and with chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) fluorochromes. All accessions analysed showed chromosome number 2n= 16, karyotype formula 6M+2SM, symmetrical karyotypes, reticulate interphase nuclei, and chromosomes with uniform chromatin condensation from prophase to metaphase. The fluorochromes staining showed differences in the amount and distribution of heterochromatin along the chromosomes and between accessions studied. Based on the distribution pattern of these small polymorphisms, it was possible to separate the seven accessions into three groups. It was also possible to differentiate some of the accessions individually. One of the results obtained showed a heteromorphic distension of the nucleolar organiser region observed on the chromosome pairs 6 or 7 with peculiar characteristics. It was suggested for example, that the heteromorphic block of heterochromatin (CMA+++/DAPI-) on chromosome 6 of the "Branco Mineiro Piauí" accession can be used as a marker to identify this genotype or may be associated with some character of economic interest.

Allium sativum L. é uma erva da família Alliaceae com sabor e aroma específicos e propriedades medicinais e nutracêuticas amplamente comercializada em diversos países. O Brasil é um dos maiores importadores de alho do mundo, apesar da sua produção ser restrita e limitada ao consumo interno. Assim, explorar a diversidade genética do alho comercial conservado em bancos de germoplasma é essencial para fornecer informações genéticas adicionais acerca dessa cultura economicamente importante. Uma ferramenta adequada para esse fim é a caracterização citogenética desses acessos. Este estudo teve como objetivo caracterizar a diversidade citogenética entre sete acessos de alho de um Banco de Germoplasma no Brasil. Os cariótipos foram obtidos por coloração convencional e com os fluorocromos de cromomicina A3 (CMA) e 4,6-diamidino-2-fenilindol (DAPI). Todos os acessos analisados apresentaram número cromossômico 2n = 16, fórmula cariotípica 6M + 2SM, cariótipos simétricos, núcleos reticulados em intérfase e cromossomos com condensação uniforme da cromatina da prófase para a metáfase. A coloração com fluorocromos mostrou diferenças na quantidade e distribuição de heterocromatina ao longo dos cromossomos e entre os acessos estudados. Com base no padrão de distribuição desses pequenos polimorfismos, foi possível separar os sete acessos em três grupos. Também foi possível diferenciar individualmente alguns dos acessos. Um dos resultados obtidos mostrou distensão heteromórfica da região organizadora nucleolar observada nos pares dos cromossomos 6 ou 7 com características peculiares. Foi sugerido, por exemplo, que o bloco heteromórfico de heterocromatina (CMA +++ / DAPI-) no cromossomo 6 do acesso “Branco Mineiro Piauí” pode ser usado como um marcador para identificar esse genótipo ou pode estar associado a algum caráter de interesse econômico.

Herbivory and leaf expansion of Cyathea phalerata Mart (Cyatheaceae) in subtropical Atlantic Forest, southern Brazil / Herbivoria e expansão foliar de Cyathea phalerata Mart. (Cyatheaceae) em Floresta Atlântica subtropical no sul do Brasil

Herbivory is an interaction with great impact on plant communities since relationships between herbivores and plants are fundamental to the distribution and abundance of species over time and space. The aim of this study was to monitor the rate of leaf expansion in the tree fern Cyathea phalerata and evaluate the damage caused by herbivores to leaves of different ages and whether such damage is related to temperature and precipitation. The study was performed in a subtropical Atlantic Forest fragment located in the municipality of Caraá, in the northeast hillside of Rio Grande do Sul state, in southern Brazil. We monitored 24 mature individuals of C. phalerata with croziers in a population of approximately 50 plants. Leaf expansion rate, percentage of damaged leaves and leaf blade consumption rate by herbivory were calculated. Monthly means for temperature and accumulated rainfall were calculated from daily data. Croziers of C. phalerata were found to expand rapidly during the first and second months after emergence (3.98 cm day-¹; 2.91 cm day-¹, respectively). Damage caused by herbivory was observed in all of the monitored leaves, but none of the plants experienced complete defoliation. The highest percentage (57%) of damaged leaves was recorded at 60 days of monitoring, and also the highest monthly consumption rate of the blade (6.04%) occurred with young, newly-expanded leaves, while this rate remained between 1.50 and 2.21% for mature leaves. Rates of monthly leaf consumption and damaged leaves showed positive and strong relationship with each other and with temperature. The rapid leaf expansion observed for C. phalerata can be considered a phenological strategy to reduce damage to young leaves by shortening the developmental period and accelerating the increase of defenses in mature leaves.

A herbivoria é uma interação de grande impacto sobre as comunidades de plantas, uma vez que as relações entre herbívoros e plantas são fundamentais para a distribuição e abundância das espécies ao longo do tempo e do espaço. O objetivo deste estudo foi monitorar a taxa de expansão foliar da samambaia arborescente Cyathea phalerata e avaliar o dano causado por herbívoros a folhas de diferentes idades, assim como verificar se este dano está relacionado à temperatura e à precipitação. O estudo foi realizado em um fragmento de Floresta Atlântica subtropical localizado no município de Caraá, na encosta nordeste do estado do Rio Grande do Sul, no sul do Brasil. Nós monitoramos 24 indivíduos maduros de C. phalerata com báculos em uma população de aproximadamente 50 plantas. A taxa de expansão foliar, a porcentagem de folhas danificadas e a taxa de consumo da lâmina foliar por herbivoria foram calculados. Médias mensais de temperatura e precipitação acumulada foram calculadas a partir de dados diários. Báculos de C. phalerata expandiram rapidamente durante o primeiro e o segundo mês após emergência (3,98 cm dia-¹; 2,91 cm dia-¹, respectivamente). O dano causado por herbivoria foi observado em todas as folhas monitoradas, mas nenhuma das plantas sofreu desfolhação completa. A maior porcentagem (57%) de folhas danificadas foi registrada aos 60 dias de monitoramento, e também a maior taxa de consumo mensal (6,04%) ocorreu em folhas jovens, recém expandidas, enquanto esta taxa permaneceu entre 1,50 e 2,21% em folhas maduras. As taxas mensais de consumo da lâmina foliar e de folhas danificadas mostraram relação positiva e forte entre si e com a temperatura. A rápida expansão foliar observada em C. phalerata pode ser considerada uma estratégia fenológica para reduzir o dano a folhas jovens, abreviando o período de desenvolvimento e acelerando o aumento das defesas em folhas maduras.

Integrative analysis in toxicological assessment of the insecticide Malathion in Allium cepa L. system

Abstract For many centuries human populations have been suffering and trying to fight with disease-bearing mosquitoes. Emerging and reemerging diseases such as Dengue, Zika, and Chikungunya affect billions of people around the world and recently has been appealing to control with chemical pesticides. Malathion (MT) is one of the main pesticides used against mosquitoes, the vectors of these diseases. This study aimed to assess cytotoxicity and mutagenicity of the malathion for the bioindicator Allium cepa L. using a multivariate and integrative approach. Moreover, an appendix table was compiled with all available literature of insecticides assessed by the Allium cepa system to support our discussion. Exposures during 48h to 0.5 mg mL-1 and 1.0 mg mL-1 MT were compared to the negative control (distilled water) and positive control (MMS solution at 10 mg L-1). The presence of chromosomal aberrations, micronuclei frequency, and mitotic index abnormalities was evaluated. Anaphase bridges were the alterations with higher incidence and presented a significantly elevated rate in the concentration of 0.5 mg mL-1, including when compared to the positive control. The integrative discriminant analysis summarizes that MT in assessed concentrations presented effects like the positive control, corroborating its potential of toxicity to DNA. Therefore, it is concluded that MT in its pure composition and in realistic concentrations used, has genotoxic potential in the biological assessment of A. cepa cells. The multivariate integrative analysis was fundamental to show a whole response of all data, providing a global view of the effect of MT on DNA.

Resumo Por muitos séculos, as populações humanas sofrem e tentam combater os mosquitos transmissores de doenças. Doenças emergentes e reemergentes como Dengue, Zika e Chikungunya afetam bilhões de pessoas em todo o mundo e, recentemente, vem apelando ao controle com pesticidas químicos. O Malation (MT) é um dos principais pesticidas usados contra mosquitos, vetores dessas doenças. O objetivo deste estudo foi avaliar a citotoxicidade e a mutagenicidade do MT para o bioindicador Allium cepa L. usando uma abordagem multivariada e integrativa. Além disso, uma tabela suplementar foi compilada com toda a literatura disponível de inseticidas avaliada pelo sistema Allium cepa para apoiar nossa discussão. Exposições ao MT durante 48h a 0,5 mg mL-1 e 1,0 mg mL-1 foram comparadas a um controle negativo (água destilada) e um controle positivo (10 mg L-1 de MMS). Foram avaliadas a presença de aberrações cromossômicas, frequência de micronúcleos e anormalidades no índice mitótico. As pontes anafásicas foram as alterações com maior incidência e apresentaram uma taxa significativamente elevada na concentração de 0,5 mg mL-1, inclusive quando comparadas ao controle positivo. A análise discriminante integrativa resume que o MT nas concentrações avaliadas apresentou efeitos semelhantes ao controle positivo, corroborando seu potencial de toxicidade para o DNA. Portanto, conclui-se que o MT, em sua composição pura e nas concentrações realistas utilizadas, possui potencial genotóxico na avaliação biológica de células de A. cepa. A análise integrativa multivariada foi fundamental para mostrar uma resposta completa de todos os dados, fornecendo uma visão global do efeito da MT no DNA.

Variations in heterochromatin content reveal important polymorphisms for studies of genetic improvement in garlic (Allium sativum L.)

Abstract Allium sativum L. is an herb of the Alliaceae family with a specific taste and aroma and medicinal and nutraceutical properties that are widely marketed in several countries. Brazil is one of the largest importers of garlic in the world, despite of its production is restricted and limited to internal consumption. Thus, explore the genetic diversity of commercial garlic conserved at germplasm banks is essential to generate additional genetic information about its economically important crop. A suitable tool for this purpose is the cytogenetic characterisation of these accessions. This study aimed to characterise the cytogenetic diversity among seven accessions of garlic from a Germplasm Bank in Brazil. The karyotypes were obtained by conventional staining and with chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) fluorochromes. All accessions analysed showed chromosome number 2n = 16, karyotype formula 6M+2SM, symmetrical karyotypes, reticulate interphase nuclei, and chromosomes with uniform chromatin condensation from prophase to metaphase. The fluorochromes staining showed differences in the amount and distribution of heterochromatin along the chromosomes and between accessions studied. Based on the distribution pattern of these small polymorphisms, it was possible to separate the seven accessions into three groups. It was also possible to differentiate some of the accessions individually. One of the results obtained showed a heteromorphic distension of the nucleolar organiser region observed on the chromosome pairs 6 or 7 with peculiar characteristics. It was suggested for example, that the heteromorphic block of heterochromatin (CMA+++/DAPI-) on chromosome 6 of the Branco Mineiro Piauí accession can be used as a marker to identify this genotype or may be associated with some character of economic interest.

Resumo Allium sativum L. é uma erva da família Alliaceae com sabor e aroma específicos e propriedades medicinais e nutracêuticas amplamente comercializada em diversos países. O Brasil é um dos maiores importadores de alho do mundo, apesar da sua produção ser restrita e limitada ao consumo interno. Assim, explorar a diversidade genética do alho comercial conservado em bancos de germoplasma é essencial para fornecer informações genéticas adicionais acerca dessa cultura economicamente importante. Uma ferramenta adequada para esse fim é a caracterização citogenética desses acessos. Este estudo teve como objetivo caracterizar a diversidade citogenética entre sete acessos de alho de um Banco de Germoplasma no Brasil. Os cariótipos foram obtidos por coloração convencional e com os fluorocromos de cromomicina A3 (CMA) e 4,6-diamidino-2-fenilindol (DAPI). Todos os acessos analisados apresentaram número cromossômico 2n = 16, fórmula cariotípica 6M + 2SM, cariótipos simétricos, núcleos reticulados em intérfase e cromossomos com condensação uniforme da cromatina da prófase para a metáfase. A coloração com fluorocromos mostrou diferenças na quantidade e distribuição de heterocromatina ao longo dos cromossomos e entre os acessos estudados. Com base no padrão de distribuição desses pequenos polimorfismos, foi possível separar os sete acessos em três grupos. Também foi possível diferenciar individualmente alguns dos acessos. Um dos resultados obtidos mostrou distensão heteromórfica da região organizadora nucleolar observada nos pares dos cromossomos 6 ou 7 com características peculiares. Foi sugerido, por exemplo, que o bloco heteromórfico de heterocromatina (CMA +++ / DAPI-) no cromossomo 6 do acesso Branco Mineiro Piauí pode ser usado como um marcador para identificar esse genótipo ou pode estar associado a algum caráter de interesse econômico.

Herbivory and leaf expansion of Cyathea phalerata Mart. (Cyatheaceae) in subtropical Atlantic Forest, southern Brazil

Abstract Herbivory is an interaction with great impact on plant communities since relationships between herbivores and plants are fundamental to the distribution and abundance of species over time and space. The aim of this study was to monitor the rate of leaf expansion in the tree fern Cyathea phalerata and evaluate the damage caused by herbivores to leaves of different ages and whether such damage is related to temperature and precipitation. The study was performed in a subtropical Atlantic Forest fragment located in the municipality of Caraá, in the northeast hillside of Rio Grande do Sul state, in southern Brazil. We monitored 24 mature individuals of C. phalerata with croziers in a population of approximately 50 plants. Leaf expansion rate, percentage of damaged leaves and leaf blade consumption rate by herbivory were calculated. Monthly means for temperature and accumulated rainfall were calculated from daily data. Croziers of C. phalerata were found to expand rapidly during the first and second months after emergence (3.98 cm day-1; 2.91 cm day-1, respectively). Damage caused by herbivory was observed in all of the monitored leaves, but none of the plants experienced complete defoliation. The highest percentage (57%) of damaged leaves was recorded at 60 days of monitoring, and also the highest monthly consumption rate of the blade (6.04%) occurred with young, newly-expanded leaves, while this rate remained between 1.50 and 2.21% for mature leaves. Rates of monthly leaf consumption and damaged leaves showed positive and strong relationship with each other and with temperature. The rapid leaf expansion observed for C. phalerata can be considered a phenological strategy to reduce damage to young leaves by shortening the developmental period and accelerating the increase of defenses in mature leaves.

Resumo A herbivoria é uma interação de grande impacto sobre as comunidades de plantas, uma vez que as relações entre herbívoros e plantas são fundamentais para a distribuição e abundância das espécies ao longo do tempo e do espaço. O objetivo deste estudo foi monitorar a taxa de expansão foliar da samambaia arborescente Cyathea phalerata e avaliar o dano causado por herbívoros a folhas de diferentes idades, assim como verificar se este dano está relacionado à temperatura e à precipitação. O estudo foi realizado em um fragmento de Floresta Atlântica subtropical localizado no município de Caraá, na encosta nordeste do estado do Rio Grande do Sul, no sul do Brasil. Nós monitoramos 24 indivíduos maduros de C. phalerata com báculos em uma população de aproximadamente 50 plantas. A taxa de expansão foliar, a porcentagem de folhas danificadas e a taxa de consumo da lâmina foliar por herbivoria foram calculados. Médias mensais de temperatura e precipitação acumulada foram calculadas a partir de dados diários. Báculos de C. phalerata expandiram rapidamente durante o primeiro e o segundo mês após emergência (3,98 cm dia-1; 2,91 cm dia-1, respectivamente). O dano causado por herbivoria foi observado em todas as folhas monitoradas, mas nenhuma das plantas sofreu desfolhação completa. A maior porcentagem (57%) de folhas danificadas foi registrada aos 60 dias de monitoramento, e também a maior taxa de consumo mensal (6,04%) ocorreu em folhas jovens, recém expandidas, enquanto esta taxa permaneceu entre 1,50 e 2,21% em folhas maduras. As taxas mensais de consumo da lâmina foliar e de folhas danificadas mostraram relação positiva e forte entre si e com a temperatura. A rápida expansão foliar observada em C. phalerata pode ser considerada uma estratégia fenológica para reduzir o dano a folhas jovens, abreviando o período de desenvolvimento e acelerando o aumento das defesas em folhas maduras.

Herbivory and leaf expansion of Cyathea phalerata Mart. (Cyatheaceae) in subtropical Atlantic Forest, southern Brazil / Herbivoria e expansão foliar de Cyathea phalerata Mart. (Cyatheaceae) em Floresta Atlântica subtropical no sul do Brasil

Abstract Herbivory is an interaction with great impact on plant communities since relationships between herbivores and plants are fundamental to the distribution and abundance of species over time and space. The aim of this study was to monitor the rate of leaf expansion in the tree fern Cyathea phalerata and evaluate the damage caused by herbivores to leaves of different ages and whether such damage is related to temperature and precipitation. The study was performed in a subtropical Atlantic Forest fragment located in the municipality of Caraá, in the northeast hillside of Rio Grande do Sul state, in southern Brazil. We monitored 24 mature individuals of C. phalerata with croziers in a population of approximately 50 plants. Leaf expansion rate, percentage of damaged leaves and leaf blade consumption rate by herbivory were calculated. Monthly means for temperature and accumulated rainfall were calculated from daily data. Croziers of C. phalerata were found to expand rapidly during the first and second months after emergence (3.98 cm day-1; 2.91 cm day-1, respectively). Damage caused by herbivory was observed in all of the monitored leaves, but none of the plants experienced complete defoliation. The highest percentage (57%) of damaged leaves was recorded at 60 days of monitoring, and also the highest monthly consumption rate of the blade (6.04%) occurred with young, newly-expanded leaves, while this rate remained between 1.50 and 2.21% for mature leaves. Rates of monthly leaf consumption and damaged leaves showed positive and strong relationship with each other and with temperature. The rapid leaf expansion observed for C. phalerata can be considered a phenological strategy to reduce damage to young leaves by shortening the developmental period and accelerating the increase of defenses in mature leaves.

Resumo A herbivoria é uma interação de grande impacto sobre as comunidades de plantas, uma vez que as relações entre herbívoros e plantas são fundamentais para a distribuição e abundância das espécies ao longo do tempo e do espaço. O objetivo deste estudo foi monitorar a taxa de expansão foliar da samambaia arborescente Cyathea phalerata e avaliar o dano causado por herbívoros a folhas de diferentes idades, assim como verificar se este dano está relacionado à temperatura e à precipitação. O estudo foi realizado em um fragmento de Floresta Atlântica subtropical localizado no município de Caraá, na encosta nordeste do estado do Rio Grande do Sul, no sul do Brasil. Nós monitoramos 24 indivíduos maduros de C. phalerata com báculos em uma população de aproximadamente 50 plantas. A taxa de expansão foliar, a porcentagem de folhas danificadas e a taxa de consumo da lâmina foliar por herbivoria foram calculados. Médias mensais de temperatura e precipitação acumulada foram calculadas a partir de dados diários. Báculos de C. phalerata expandiram rapidamente durante o primeiro e o segundo mês após emergência (3,98 cm dia-1; 2,91 cm dia-1, respectivamente). O dano causado por herbivoria foi observado em todas as folhas monitoradas, mas nenhuma das plantas sofreu desfolhação completa. A maior porcentagem (57%) de folhas danificadas foi registrada aos 60 dias de monitoramento, e também a maior taxa de consumo mensal (6,04%) ocorreu em folhas jovens, recém expandidas, enquanto esta taxa permaneceu entre 1,50 e 2,21% em folhas maduras. As taxas mensais de consumo da lâmina foliar e de folhas danificadas mostraram relação positiva e forte entre si e com a temperatura. A rápida expansão foliar observada em C. phalerata pode ser considerada uma estratégia fenológica para reduzir o dano a folhas jovens, abreviando o período de desenvolvimento e acelerando o aumento das defesas em folhas maduras.

Integrative analysis in toxicological assessment of the insecticide Malathion in Allium cepa L. system / Análise integrativa na avaliação toxicológica do inseticida Malation no sistema Allium cepa L

Abstract For many centuries human populations have been suffering and trying to fight with disease-bearing mosquitoes. Emerging and reemerging diseases such as Dengue, Zika, and Chikungunya affect billions of people around the world and recently has been appealing to control with chemical pesticides. Malathion (MT) is one of the main pesticides used against mosquitoes, the vectors of these diseases. This study aimed to assess cytotoxicity and mutagenicity of the malathion for the bioindicator Allium cepa L. using a multivariate and integrative approach. Moreover, an appendix table was compiled with all available literature of insecticides assessed by the Allium cepa system to support our discussion. Exposures during 48h to 0.5 mg mL-1 and 1.0 mg mL-1 MT were compared to the negative control (distilled water) and positive control (MMS solution at 10 mg L-1). The presence of chromosomal aberrations, micronuclei frequency, and mitotic index abnormalities was evaluated. Anaphase bridges were the alterations with higher incidence and presented a significantly elevated rate in the concentration of 0.5 mg mL-1, including when compared to the positive control. The integrative discriminant analysis summarizes that MT in assessed concentrations presented effects like the positive control, corroborating its potential of toxicity to DNA. Therefore, it is concluded that MT in its pure composition and in realistic concentrations used, has genotoxic potential in the biological assessment of A. cepa cells. The multivariate integrative analysis was fundamental to show a whole response of all data, providing a global view of the effect of MT on DNA.

Resumo Por muitos séculos, as populações humanas sofrem e tentam combater os mosquitos transmissores de doenças. Doenças emergentes e reemergentes como Dengue, Zika e Chikungunya afetam bilhões de pessoas em todo o mundo e, recentemente, vem apelando ao controle com pesticidas químicos. O Malation (MT) é um dos principais pesticidas usados ​​contra mosquitos, vetores dessas doenças. O objetivo deste estudo foi avaliar a citotoxicidade e a mutagenicidade do MT para o bioindicador Allium cepa L. usando uma abordagem multivariada e integrativa. Além disso, uma tabela suplementar foi compilada com toda a literatura disponível de inseticidas avaliada pelo sistema Allium cepa para apoiar nossa discussão. Exposições ao MT durante 48h a 0,5 mg mL-1 e 1,0 mg mL-1 foram comparadas a um controle negativo (água destilada) e um controle positivo (10 mg L-1 de MMS). Foram avaliadas a presença de aberrações cromossômicas, frequência de micronúcleos e anormalidades no índice mitótico. As pontes anafásicas foram as alterações com maior incidência e apresentaram uma taxa significativamente elevada na concentração de 0,5 mg mL-1, inclusive quando comparadas ao controle positivo. A análise discriminante integrativa resume que o MT nas concentrações avaliadas apresentou efeitos semelhantes ao controle positivo, corroborando seu potencial de toxicidade para o DNA. Portanto, conclui-se que o MT, em sua composição pura e nas concentrações realistas utilizadas, possui potencial genotóxico na avaliação biológica de células de A. cepa. A análise integrativa multivariada foi fundamental para mostrar uma resposta completa de todos os dados, fornecendo uma visão global do efeito da MT no DNA.

Variations in heterochromatin content reveal important polymorphisms for studies of genetic improvement in garlic (Allium sativum L. ) / Conteúdo de heterocromatina revela polimorfismos importantes para o melhoramento genético do alho (Allium sativum L. )

Abstract Allium sativum L. is an herb of the Alliaceae family with a specific taste and aroma and medicinal and nutraceutical properties that are widely marketed in several countries. Brazil is one of the largest importers of garlic in the world, despite of its production is restricted and limited to internal consumption. Thus, explore the genetic diversity of commercial garlic conserved at germplasm banks is essential to generate additional genetic information about its economically important crop. A suitable tool for this purpose is the cytogenetic characterisation of these accessions. This study aimed to characterise the cytogenetic diversity among seven accessions of garlic from a Germplasm Bank in Brazil. The karyotypes were obtained by conventional staining and with chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) fluorochromes. All accessions analysed showed chromosome number 2n = 16, karyotype formula 6M+2SM, symmetrical karyotypes, reticulate interphase nuclei, and chromosomes with uniform chromatin condensation from prophase to metaphase. The fluorochromes staining showed differences in the amount and distribution of heterochromatin along the chromosomes and between accessions studied. Based on the distribution pattern of these small polymorphisms, it was possible to separate the seven accessions into three groups. It was also possible to differentiate some of the accessions individually. One of the results obtained showed a heteromorphic distension of the nucleolar organiser region observed on the chromosome pairs 6 or 7 with peculiar characteristics. It was suggested for example, that the heteromorphic block of heterochromatin (CMA+++/DAPI-) on chromosome 6 of the "Branco Mineiro Piauí" accession can be used as a marker to identify this genotype or may be associated with some character of economic interest.

Resumo Allium sativum L. é uma erva da família Alliaceae com sabor e aroma específicos e propriedades medicinais e nutracêuticas amplamente comercializada em diversos países. O Brasil é um dos maiores importadores de alho do mundo, apesar da sua produção ser restrita e limitada ao consumo interno. Assim, explorar a diversidade genética do alho comercial conservado em bancos de germoplasma é essencial para fornecer informações genéticas adicionais acerca dessa cultura economicamente importante. Uma ferramenta adequada para esse fim é a caracterização citogenética desses acessos. Este estudo teve como objetivo caracterizar a diversidade citogenética entre sete acessos de alho de um Banco de Germoplasma no Brasil. Os cariótipos foram obtidos por coloração convencional e com os fluorocromos de cromomicina A3 (CMA) e 4,6-diamidino-2-fenilindol (DAPI). Todos os acessos analisados ​​apresentaram número cromossômico 2n = 16, fórmula cariotípica 6M + 2SM, cariótipos simétricos, núcleos reticulados em intérfase e cromossomos com condensação uniforme da cromatina da prófase para a metáfase. A coloração com fluorocromos mostrou diferenças na quantidade e distribuição de heterocromatina ao longo dos cromossomos e entre os acessos estudados. Com base no padrão de distribuição desses pequenos polimorfismos, foi possível separar os sete acessos em três grupos. Também foi possível diferenciar individualmente alguns dos acessos. Um dos resultados obtidos mostrou distensão heteromórfica da região organizadora nucleolar observada nos pares dos cromossomos 6 ou 7 com características peculiares. Foi sugerido, por exemplo, que o bloco heteromórfico de heterocromatina (CMA +++ / DAPI-) no cromossomo 6 do acesso "Branco Mineiro Piauí" pode ser usado como um marcador para identificar esse genótipo ou pode estar associado a algum caráter de interesse econômico.

Recombinant human TIM-3 ectodomain expressed in bacteria and recovered from inclusion bodies as a stable and active molecule

Introduction: Microbial systems, such as Escherichia coli, as host recombinant expression is the most versatile and the cheapest system for protein production, however, several obstacles still remain, such as recovery of soluble and functional proteins from inclusion bodies, elimination of lipopolysaccharides (LPS) contamination, incomplete synthesis, degradation by proteases, and the lack of post-translational modifications, which becomes even more complex when comes to membrane proteins, because they are difficult not only to produce but also to keep in solution in its active state. T-cell Immunoglobulin and Mucin domain 3 (TIM-3) is a type I transmembrane protein that is predominantly expressed on the surface of T lymphocytes, natural killer (NK) cells, dendritic cells, and macrophages, playing a role as a negative immune checkpoint receptor. TIM-3 comprises a single ectodomain for interaction with immune system soluble and cellular components, a transmembrane domain, and a cytoplasmic tail, responsible for the binding of signaling and scaffolding molecules. TIM-3 pathway holds potential as a therapeutic target for immunotherapy against tumors, autoimmunity, chronic virus infections, and various malignancies, however, many aspects of the biology of this receptor are still incompletely understood, especially regarding its ligands. Methods: Here we overcome, for the first time, the challenge of the production of active immune checkpoint protein recovered from bacterial cytoplasmic inclusion bodies, being able to obtain an active, and non-glycosylated TIM-3 ectodomain (TIM-3-ECD), which can be used as a tool to better understand the interactions and roles of this immune checkpoint. The TIM-3 refolding was obtained by the association of high pressure and alkaline pH. Results: The purified TIM-3-ECD showed the correct secondary structure and was recognized from anti-TIM-3 structural-dependent antibodies likewise commercial TIM-3-ECD was produced by a mammal cells system. Furthermore, immunofluorescence showed the ability of TIM-3-ECD to bind to the surface of lung cancer A549 cells and to provide an additional boost for the expression of the lymphocyte activation marker CD69 in anti-CD3/CD28 activated human PBMC. Discussion: Taken together these results validated a methodology able to obtain active checkpoint proteins from bacterial inclusion bodies, which will be helpful to further investigate the interactions of this and others not yet explored immune checkpoints.